Lysosomal degradation of alpha-synuclein in vivo

Abstract

Pathologic accumulation of alpha-synuclein is a feature of human parkinsonism and other neurodegenerative diseases. This accumulation may be counteracted by mechanisms of protein degradation that have been investigated in vitro but remain to be elucidated in animal models. In this study, lysosomal clearance of alpha-synuclein in vivo was indicated by the detection of alpha-synuclein in the lumen of lysosomes isolated from the mouse midbrain. When neuronal alpha-synuclein expression was enhanced as a result of toxic injury (i.e. treatment of mice with the herbicide paraquat) or transgenic protein overexpression, the intralysosomal content of alpha-synuclein was also significantly increased. This effect was paralleled by a marked elevation of the lysosome-associated membrane protein type 2A (LAMP-2A) and the lysosomal heat shock cognate protein of 70 kDa (hsc70), two essential components of chaperone-mediated autophagy (CMA). Immunofluorescence microscopy revealed an increase in punctate (lysosomal) LAMP-2A staining that co-localized with alpha-synuclein within nigral dopaminergic neurons of paraquat-treated and alpha-synuclein-overexpressing animals. The data provide in vivo evidence of lysosomal degradation of alpha-synuclein under normal conditions and, quite importantly, under conditions of enhanced protein burden. In the latter, increased lysosomal clearance of alpha-synuclein was mediated, at least in part, by CMA induction. It is conceivable that these neuronal mechanisms of protein clearance play an important role in neurodegenerative processes characterized by abnormal alpha-synuclein buildup.

FIGURE 1.

α-Synuclein association with lysosomes is enhanced in the midbrain of paraquat-treated mice. A, lysosomes were isolated from the midbrain of animals sacrificed 3 days after a single intraperitoneal injection of either saline or 10 mg/kg paraquat (PQ). After isolation, they were untreated or incubated with proteinase K (PK). B, lysosomes from the midbrain of saline- and paraquat-treated mice were subjected to hypotonic shock and ultracentrifugation for separation of lysosomal matrices and membranes. A and B, α-synuclein content was measured by Western blot analysis. To ensure equal loading of the gels, immunoblots were probed with an antibody against LAMP-1 (a lysosomal transmembrane protein) or cathepsin B (a marker of lysosomal matrix). Results of the densitometric quantification are the means + S.E. (n = 3) and are expressed as folds of the control (con) value in saline-treated mice. C, homogenate (postnuclear fraction) and lysosomal (membrane and matrix fractions) samples (10 μg) from the midbrain of saline- and PQ-injected mice were assayed in parallel by Western blotting using an anti-α-synuclein antibody. A representative blot is shown. Results of the densitometric quantification are the means + S.E. (n = 3) and are expressed as the ratio of lysosomal matrix/homogenate α-synuclein in control and PQ-treated animals, respectively. *, p < 0.01 versus the corresponding control group.

FIGURE 2.

Lysosomal degradation of α-synuclein. Levels of α-synuclein were measured by Western blot analysis at time 0 and after a 5-min incubation of lysosomes from the midbrain of saline- and PQ-injected mice. After isolation, lysosomes were subjected to hypotonic shock. Results of the densitometric quantification are the means + S.E. of triplicate samples and are expressed as folds of the control (con) value at time 0 in saline-treated mice. Differences in α-synuclein levels (means + S.E.) were calculated by subtracting values at 5 min from the corresponding values at time 0 in preparations from control (saline-injected) and paraquat-treated animals. *, p < 0.005 versus the corresponding control group.

FIGURE 3.

Paraquat-induced LAMP-2A up-regulation. A, lysosomal membranes were isolated from the midbrain of mice sacrificed 3 days after a single injection of either saline or PQ. Levels of LAMP-2A were measured by Western blot analysis. Blots were probed with an antibody against LAMP-1 to ensure equal loading. B, mRNA levels of LAMP-2A, LAMP-2B, and LAMP-2C were assayed by qPCR in homogenates from the ventral mesencephalon of saline and paraquat-treated mice. A and B, results in the graphs are the means + S.E. of triplicate samples and are expressed as folds of the corresponding control value in saline-treated mice. *, p < 0.01 versus the corresponding control group. C, lamp-2a transcript was amplified by PCR and separated by agarose gel electrophoresis in samples from the ventral mesencephalon of saline and paraquat-injected mice.

FIGURE 4.

Enhanced LAMP-2A immunoreactivity and LAMP-2A/α-synuclein co-localization within nigral neurons from paraquat-treated mice. A–F, midbrain sections from saline- (A–C) and paraquat- (D–F) injected mice were immunostained with antibodies against α-synuclein (A and D) and LAMP-2A (B and E). Merged images are shown in C and F. Scale bar, 20 μm. G–I, higher magnification images of a representative neuron from the substantia nigra of a paraquat-treated mouse show a punctate pattern of immunostaining for both α-synuclein (G) and LAMP-2A (H) with substantial co-localization of the two proteins (I). Scale bar equals 10 μm.

FIGURE 5.

Paraquat-induced increase in LAMP-2A and α-synuclein immunolabeling within dopaminergic neurons. A–H, midbrain sections from saline- (A–D) and paraquat- (E–H) injected mice were triple-immunolabeled with antibodies against TH (A and E), α-synuclein (B and F), and LAMP-2A (C and G). Merged images of α-synuclein and Lamp-2A immunoreactivity are shown in D and H. Representative images show that, after paraquat treatment, labeling for α-synuclein and Lamp-2A was increased within dopaminergic (TH-positive) neurons. Scale bar equals 10 μm.

FIGURE 6.

Paraquat-induced changes in hsc70. A, lysosomes were isolated from the midbrain of animals sacrificed 3 days after a single intraperitoneal injection of saline or PQ. Levels of hsc70 were measured by Western blot analysis. Blots were probed with an antibody against LAMP-1 to ensure equal loading. B, cytosolic fractions of the mouse ventral mesencephalon were immunoprecipitated with an hsc70 antibody, and eluates were then assayed for hsc70 and α-synuclein by Western blot analysis. Immunoprecipitation conditions were adjusted to yield similar levels of hsc70 in tissue eluates from control and paraquat-exposed mice. Results of the densitometric quantification are the means + S.E. of triplicate samples and are expressed as folds of the control (con) value in saline-treated mice. *, p < 0.01 versus the corresponding control group.

FIGURE 7.

Increase in LAMP-2A mRNA in transgenic mice overexpressing α-synuclein. A–C, mRNA levels of α-synuclein (α-syn), LAMP-2A, and LAMP-2B were assayed by qPCR in homogenates from the ventral mesencephalon (A), hippocampus (B), and frontal cortex (C) of control and transgenic mice. Data are the means + S.E. of quadruplicate samples and are expressed as folds of the corresponding control value. *, p < 0.0001 versus the corresponding control group. D, correlation plot for α-synuclein and LAMP-2A mRNAs in the three brain regions of the transgenic mice. R² = 0.991.

FIGURE 8.

Enhanced LAMP-2A labeling in the substantia nigra of α-synuclein-overexpressing mice. A–F, midbrain sections from control (A–C) and transgenic (D–F) mice were immunostained with antibodies against α-synuclein (A and D) and LAMP-2A (B and E). Representative images show increased labeling for both α-synuclein and LAMP-2A in transgenics and substantial co-localization of the two proteins (C and F). G–J, a second set of midbrain sections from transgenic animals was triple-labeled with antibodies recognizing TH (G), α-synuclein (H), and LAMP-2A (I). Merged image of α-synuclein and Lamp-2A immunoreactivity is shown in J. Scale bars equal 15 μm.

FIGURE 9.

Increase in cortical LAMP-2A in α-synuclein-overexpressing mice. Levels of LAMP-2A were measured by Western blot analysis in homogenates from the frontal cortex of control and transgenic mice. Blots were probed with an antibody against β-actin to ensure equal loading. Results of the densitometric quantification are the means + S.E. of triplicate samples and are expressed as folds of the value in control mice. *, p < 0.001 versus the control group.

FIGURE 10.

Increased hsc70 immunolabeling in the substantia nigra of α-synuclein-overexpressing mice. A–F, midbrain sections from control (A–C) and transgenic (D–F) mice were immunostained with antibodies against α-synuclein (A and D) and hsc70 (B and E). Merged images are shown in C and F. Representative images of the substantia nigra show increased immunolabeling for α-synuclein and hsc70 and neuronal co-localization of the two proteins in transgenic mice. Scale bar equals 10 μm.